JP2020509747A - Dimer avoidance multiplex polymerase chain reaction for amplifying multiple targets - Google Patents

Abstract

The present disclosure relates to nucleic acid amplification methods that avoid problems with primer dimer formation. This method is referred to herein as dimer avoidance multiplex polymerase chain reaction (dam-PCR). The methods disclosed herein generally include the following steps: reverse transcribing at least one DNA first strand, eg, cDNA, from an RNA sample, wherein each DNA first strand has a reverse common primer binding site. Is incorporated; a step of selecting each DNA first strand; a step of synthesizing at least one DNA second strand from each of said at least one DNA first strand, wherein each DNA second strand is forwarded Incorporating a common primer binding site; selecting each cDNA second strand; and amplifying the DNA strand with a common primer. Alternatively, the method may be performed using a gDNA template. The method described herein avoids primer dimer formation by selection of DNA strands and removal of unused primers prior to amplification, allowing for much higher sensitivity and efficiency compared to traditional multiplex PCR methods. To do.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application entitled, filed March 9, 2017 "dimer avoiding multiplex polymerase chain reaction for amplifying a plurality of target (Dimer Avoided Multiplex Polymerase Chain Reaction for Amplification of Multiple Targets) " No. 62 / 469,309, which is hereby incorporated herein by reference.

Related Art The polymerase chain reaction (PCR) has made it possible to use DNA amplification in a variety of applications, including molecular diagnostic tests. Multiplex PCR, which amplifies multiple nucleic acids in a sample, has been developed to enable detection and identification of multiple target sequences. In multiplex PCR, multiple primers must be selected that specifically bind to different target sequences so that they can be amplified. However, the use of multiple primers has proven to be problematic under conventional multiplex PCR methodologies that require optimization of primer sets, temperature conditions, and enzymes to meet different conditions that may be needed for different primers. I have. Therefore, considerable planning and testing may be required to find primer sets compatible with conventional multiplex PCR methods.

  The difficulties associated with multiplex PCR are further exacerbated by the formation of primer dimers, which tends to occur when multiple primers are used. The formation of primer dimers can result in some target sequences amplifying very efficiently, while others amplify very inefficiently or not at all. This potential for amplification also makes it difficult or impossible to accurately perform the final quantitative analysis, and also determines which primers can be combined successfully in a particular multiplex PCR assay. Significant primer optimization is required to determine. The problem of the primer dimer may also involve a method in which the target sequence is concentrated using gene-specific primers in the first PCR cycle, and then further amplified using common primers. Nevertheless, the current paradigm states that primer selection must be optimized to achieve the ideal performance of multiplex PCR [eg Canzar, et al. Bioinformatics, 2016, 1-3 (Non-Patent Document 1) (“Canzar”)].

  Therefore, there remains a need for a method that can amplify multiple target sequences using multiple primers while avoiding the negative effects of primer dimer formation without the need for cumbersome and costly primer optimization. I do.

Canzar, et al. Bioinformatics, 2016, 1-3

  In one aspect, the disclosure relates to a method comprising the steps of: reverse transcripting at least one cDNA first strand from a mRNA containing at least one target sequence using a reverse primer mix to form a first strand cDNA. Wherein the reverse primer mix comprises at least one reverse primer configured to incorporate a reverse common primer binding site into each cDNA first strand. Selecting; from each of the at least one cDNA first strand, synthesizing at least one cDNA second strand using a forward primer mix to form at least one first strand: second strand complex. The forward primer mix contains at least one forward primer, wherein each forward primer binds to a specific cDNA first strand. Selecting each cDNA second strand; selecting each first strand: second strand complex, and incorporating the forward common primer binding site into each cDNA second strand. Amplifying a cDNA strand with a reverse common primer that binds to the at least one reverse common primer binding site and with a forward common primer that binds to the at least one forward common primer binding site; and Selecting the cDNA strand obtained. In certain embodiments, the method comprises using the reverse common primer to bind to the at least one reverse common primer binding site and using the forward common primer to bind to the at least one forward common primer binding site. The method further includes the step of amplifying the cDNA strand. In certain embodiments of the method, the reverse primer mix comprises at least one reverse primer, wherein the at least one reverse primer comprises additional nucleotides that are incorporated as identification markers into each first cDNA strand. In certain aspects of the method, the forward primer mix comprises at least one forward primer, wherein the at least one forward primer comprises additional nucleotides that are incorporated as identification markers into each second cDNA strand. In certain embodiments of the method, each selection comprises separating the cDNA strand from the primer mix using magnetic beads. In certain embodiments of the method, each selection comprises separating the cDNA strand from the primer mix by column purification. In certain aspects of the method, each selection comprises enzymatic cleavage of the primer mix. In certain embodiments, the first strand cDNA comprises a first strand cDNA: RNA complex. In certain aspects, the disclosure relates to a method of diagnosing the presence of a disease in a subject, the method comprising: providing a sample from the subject, wherein the sample contains a disease factor. Suspecting that the disease factor is characterized by a target sequence; performing the method described in this paragraph on the nucleic acid in the sample; sequencing the amplified DNA strand; and the disease Detecting the target sequence from the agent. In certain aspects, the disclosure relates to a method of generating an immune status profile in a subject, the method comprising performing the method described in this paragraph on nucleic acid from a sample of leukocytes from the subject; Sequencing the DNA strands; and identifying and quantifying one or more DNA sequences representing T cell receptors, antibodies, and MHC rearrangements to generate an immune status profile for the subject. In certain embodiments, the mRNA is obtained from a single cell.

  In certain aspects, the disclosure relates to a method comprising the steps of: synthesizing at least one DNA first strand from a genomic DNA containing at least one target sequence using a first primer mix; : Forming a DNA complex, wherein the first primer mix contains at least one first primer, each first primer binding to a specific target sequence and a first common primer binding Selecting a site from each of the at least one DNA first strands using a second primer mix from each of the at least one DNA first strands. Synthesizing at least one DNA second strand to form a first strand: second strand complex, wherein the second primer mix comprises at least one second primer, wherein each second primer is Binds to a specific DNA first strand And a second common primer binding site is incorporated into each DNA second strand; selecting each first strand: second strand complex; the at least one first common primer binding site Amplifying a DNA strand using a first common primer that binds to the at least one second common primer and binding to the at least one second common primer binding site; and selecting the amplified DNA strand . In certain embodiments, the genomic DNA is obtained from a single cell. In certain aspects, the disclosure relates to a method of diagnosing the presence of a disease in a subject, the method comprising: providing a sample from the subject, wherein the sample contains a disease factor. Suspecting that the disease factor is characterized by a target sequence; isolating the nucleic acid from the sample; performing the method described in this paragraph on the isolated nucleic acid; amplified. Sequencing a DNA strand; and detecting a target sequence from the disease factor. In certain aspects, the disclosure relates to a method of generating an immune status profile in a subject, comprising: performing the method described in this paragraph on nucleic acid from a sample of leukocytes from the subject. Sequencing the amplified DNA strands; and identifying and quantifying one or more DNA sequences representing T cell receptors, antibodies, and MHC rearrangements to generate an immune status profile for the subject Stage to do.

  In certain aspects of the method, the first primer mix is a reverse primer mix, each first primer is a reverse primer, each first common primer is a reverse common primer, and each first common primer binding site is a reverse common primer. The second primer mix is a forward primer mix, each second primer is a forward primer, each second common primer is a forward common primer, and each second common primer binding site is a forward common primer. Binding site. In certain embodiments of the method, the first primer mix is a forward primer mix, each first primer is a forward primer, each first common primer is a forward common primer, and each first common primer binding site is a forward common primer. The second primer mix is a reverse primer mix, each second primer is a reverse primer, each second common primer is a reverse common primer, and each second common primer binding site is a reverse common primer. Binding site. In certain embodiments of the method, the first primer mix is a primer mix comprising at least one forward and at least one reverse primer, wherein each first primer is a forward or reverse primer, and each first common primer is A forward or reverse common primer, wherein each first common primer binding site is a forward or reverse common primer binding site; the second primer mix comprises at least one forward and at least one reverse primer, wherein the second Both the forward and reverse primers in the primer mix are not included in the first primer mix, each second common primer is a forward or reverse common primer, and each second common primer binding site is Forward or reverse common primer binding site. In certain embodiments, the method comprises using the reverse common primer to bind to at least one reverse common primer binding site and using the forward common primer to bind to at least one forward common primer binding site, The method further includes the step of amplifying. In certain aspects of the method, the first primer mix comprises primers that include additional nucleotides that are incorporated as identification markers into each first DNA strand. In certain embodiments of the method, the second primer mix includes primers that include additional nucleotides that are incorporated as identification markers into each second DNA strand. In certain aspects of the method, each selection comprises separating the DNA strands from the primer mix using magnetic beads. In certain aspects of the method, each selection comprises separating the DNA strands from the primer mix by column purification. In certain aspects of the method, each selection comprises enzymatic cleavage of the primer mix.

  The present disclosure can be better understood with reference to the following drawings. The scale of the elements in the figures is not necessarily relative to each other, but instead highlights the principles of the present disclosure. Further, like reference numerals indicate corresponding parts throughout several drawings.

1A-1C illustrate steps of an exemplary embodiment of a dam-PCR methodology using an RNA template. FIG. 1A shows a single cycle of first strand cDNA synthesis (first strand tagging) by reverse transcription, removal of unused reverse primer mix, and selection of first strand cDNA. 1A-1C illustrate steps of an exemplary embodiment of a dam-PCR methodology using an RNA template. FIG. 1B shows the steps of single cycle second strand DNA synthesis (second strand tagging), removal of unused forward primer mix, and selection of first strand: second strand DNA duplex. 1A-1C illustrate steps of an exemplary embodiment of a dam-PCR methodology using an RNA template. FIG. 1C shows the step of amplifying the target using common primers and the optional step of repeating the selection and amplification. 2A-2C illustrate steps of an exemplary embodiment of a dam-PCR methodology using a genomic DNA ("gDNA") template. FIG. 2A shows the steps of first strand DNA synthesis (first strand tagging) in the first primer mix, removal of unused first primer mix, and selection of first strand: DNA duplex. 2A-2C illustrate steps of an exemplary embodiment of a dam-PCR methodology using a genomic DNA ("gDNA") template. FIG. 2B shows the steps of second strand DNA synthesis and removal of unused second primer mix. 2A-2C illustrate steps of an exemplary embodiment of a dam-PCR methodology using a genomic DNA ("gDNA") template. FIG. 2C shows the step of amplifying the target using common primers and the optional step of repeating the selection and amplification. 3A and 3B illustrate the effect of primer dimer formation on output. FIG. 3A is a gel showing the predominance of primer dimer formation when arm-PCR is used on single cells, and the corresponding decrease in main product band intensity due to primer dimer formation. The percentage on the gel indicates the percentage of the number of sequencing reads wasted by sequencing of the primer dimer product remaining after the final library cleanup, and was obtained by analyzing next-generation sequencing data of each cell data. Each lane represents a unique single cell after amplification with an arm-PCR multiplex mix covering the alpha and beta TCR loci and additional phenotypic markers. 3A and 3B illustrate the effect of primer dimer formation on output. FIG.3B is a table showing the likelihood of primer-dimer formation between primer pairs during PCR in a mix containing both forward and reverse primers, and the number of wasted reads as a result of primer-dimer formation. May account for as much as 31% of the intended number of reads for a sample. 4A and 4B illustrate primer dimer formation with as little as one base pair overlap. Figure 4A shows that arm-PCR was performed in the absence of template under several cycling conditions, including RT-single cell protocol (scRT), no-RT single cell protocol (scPCR), and iR-10-10 protocol (iR1010) FIG. 4 is a gel showing intentional primer dimer formation by the following method. Lanes 1-3 represent primer dimer formation of one nucleotide overlap, which is formed between primer iTRAV_2 and primer iTRAC, despite using the protocol. Lanes 4-6 represent primer dimer formation of a 4 nucleotide overlap between primer iTRAV_2 and primer IL-17F-Ri-09, which is formed despite using the protocol. Lanes 6-9 show primer dimer formation of a 6 nucleotide overlap between primer iTRAV_2 and primer BCL6Ri_MD_11, which is formed despite using the protocol. Lanes 10-12 represent primer dimer formation of a 6 nucleotide overlap between primer iTRAV_40 and primer BCL6Ri_MD_11, which is formed despite using the protocol. 4A and 4B illustrate primer dimer formation with as little as one base pair overlap. FIG. 4B illustrates specifically overlapping base pairs in a particular primer dimer. The ranks of the primer-dimer products of the sequencing results of the single cells amplified by arm-PCR shown in FIGS.3A and 3B are also provided, with `` Top 1 '' indicating the highest ranked primer-dimer frequency in the NGS sequencing results. "Top 2" represents the second highest primer dimer frequency among NGS sequencing results, and so on. FIG. 5 is a gel showing that a pair of primers with high primer dimer propensity can eliminate amplification of the desired product band. In lane 6 of the illustrated agarose gel, four primers for amplifying the target IL-10 were successfully included in the PCR. Addition of T-bet_Fi eliminates amplification of the band of interest, as shown in lane 7. Similarly, a multiplex primer mix covering the 4 primers of T-bet and containing IL-17A-Fo successfully amplifies the target, as shown in lane 9. However, the addition of one harmful primer, the FoxP3Ri reverse inner primer, eliminates amplification of the main product band, as shown in lane 10. FIG. 6 is a gel showing that adjusting the annealing temperature does not eliminate the primer dimer effect. Four different primer pairs known to form primer dimers were tested at several annealing temperatures from 59.9 ° C. to 66 ° C. to eliminate primer dimer formation, but were not successful. FIGS. 7A and 7B are gels illustrating the effect of primer-dimer formation of arm-PCR versus dam-PCR. FIG. 7A is a gel showing that dam-PCR overcomes the inhibitory primer dimer effect by bead selection. Lanes 1-2 are arm-PCR controls. Lanes 3-4 are arm-PCR controls with a pair of primer spike-ins known to be responsible for dimerization. Lanes 5-6 are single cycle dam-PCR without spike-in of the primer dimer pair. Lanes 7-8 are single cycle dam-PCR with spike-in of the primer dimer pair. Lanes 9-10 are dam-PCR with linear amplification without spike-in of the primer dimer pair. Lanes 11-12 are dam-PCR with linear amplification with spike-in of the primer dimer pair. Lane 13 is the negative control. FIGS. 7A and 7B are gels illustrating the effect of primer-dimer formation of arm-PCR versus dam-PCR. Figure 7B shows that dam-PCR replaces bead selection after the first round of amplification using common forward and reverse primers for dam-PCR, or after the first round of RT-PCR for arm-PCR. Fig. 9 is a gel showing that the inhibitory primer-dimer effect is overcome by transfer. Lanes 1-2 are arm-PCR controls. Lanes 3-4 are arm-PCR controls with a pair of primer spike-ins known to be responsible for dimerization. Lanes 5-6 are single cycle dam-PCR without spike-in of the primer dimer pair. Lanes 7-8 are single cycle dam-PCR with spike-in of the primer dimer pair. Lanes 9-10 are dam-PCR with linear amplification without spike-in of the primer dimer pair. Lanes 11-12 are dam-PCR with linear amplification with spike-in of the primer dimer pair. Lane 13 is the negative control. FIG. 8 is a gel comparing single cell amplification by dam-PCR versus arm-PCR. Lanes 1-6 represent single cells amplified by dam-PCR, and lanes 7-14 represent single cells amplified by arm-PCR. Cells amplified by dam-PCR have similar endpoint strength and no primer dimer, but cells amplified by arm-PCR show diversity in endpoint PCR and Shows amplification. FIG. 9 is a gel showing the ability of the selection step to remove unused primer. The lane labeled "standard curve" was used to evaluate the amount of primer carryover between first strand tagging to second strand tagging and between second strand tagging to amplification. Lanes 1 and 2 represent two rounds of magnetic bead selection, removing more than 99.99% of unused primer compared to the standard curve. Lanes 3 and 4 represent a single magnetic bead selection step that removes more than 99.9% of unused primer compared to the standard curve. FIGS. 10A-10B are gels showing dam-PCR of gDNA using various multiplex primer mixes. FIG. 10A shows arm-PCR (lanes 1-4) versus dam-PCR (lanes 5-9) for TCR beta locus amplification. FIGS. 10A-10B are gels showing dam-PCR of gDNA using various multiplex primer mixes. FIG. 10B shows arm-PCR versus dam-PCR with a two-input tumor multiplex panel of gDNA. Lanes 1-7 represent 200 ng of gDNA input, lanes 1-3 were performed by arm-PCR, and lanes 4-7 were performed by dam-PCR. Lanes 8-14 represent a 540 ng input, lanes 8-10 were performed with arm-PCR, and lanes 11-14 were performed with dam-PCR. FIG. 11 shows a typical gDNA multiplex PCR using primers designed to cover two exons. This type of design introduces duplication between the two amplification products so that the gene can be reconstructed during the bioinformatics process. Such methods produce non-target amplification due to the compatibility of additional primers required for the generation of overlap, resulting in shorter PCR products that compete. FIG. 12 is a diagram showing dam-PCR of gDNA and the merits associated with dam-PCR compared to ordinary multiplex PCR. Specifically, during the first cycle of tagging, a set of primers covering a larger exon product can be used. After cleanup of the first primer mix, the second primer mix includes an inner primer that interacts only with the respective first strand product. Since these primers do not participate in any amplification and are used only once during tagging, no shorter competing PCR products are generated. FIG. 13 is an agarose gel comparison of the arm-PCR multiplex PCR strategy and the dam-PCR strategy covering long gDNA gene targets with overlap, specifically covering HLA targets. Above the agarose gel, a diagram is provided that clearly shows the location of the primers referenced in the agarose gel. Lane 1 shows the amplification pattern when only gene target A was amplified using arm-PCR. Lane 2 shows the amplification pattern when only gene target B was amplified using arm-PCR. Lane 3 shows the amplification pattern of one possible off-target amplification from the interaction of T2 forward and T1 reverse by deliberately creating overlapping segments. Lane 4 shows the long amplification products of the T1 forward and T2 reverse primers. Lanes 5-6 show arm-PCR from the complete multiplex mix. This result is mostly dominated by less desirable, short, off-target products. Target A only gene target (produced from T1 forward and T1 reverse), target B only gene target (produced from T2 forward and T2 reverse), and target A (produced from T1 forward and T2 reverse) Gene targets, both Target B and Target B, are smeared and almost absent due to the short product competing for DNA polymerase activity with the desired product. Lane 7 shows the amplification pattern when only gene target A was amplified using dam-PCR with T1 reverse during first cycle tagging and T1 forward during second cycle tagging. Lane 8 shows the amplification pattern when only gene target B was amplified using dam-PCR with T2 reverse during first cycle tagging and T2 forward during second cycle tagging. Lanes 9-10 show the results of a fully multiplexed dam-PCR strategy. In the first round of tagging, a longer first strand product was generated using T1 forward and T2 reverse. In the second round of tagging, T1 reverse and T2 forward interacted independently with the first strand product generated during first strand tagging to synthesize the second strand. After second strand tagging, cleanup, and amplification with a pair of primers common to the first round of targets, there were no competing short products and amplification of the desired product was obtained.

DETAILED DESCRIPTION The present disclosure relates to nucleic acid amplification methods that avoid problems with primer dimer formation. This method is referred to herein as dimer avoidance multiplex polymerase chain reaction (dam-PCR). The method disclosed herein generally comprises the steps of: reverse transcription of at least one DNA first strand, eg, cDNA, from an RNA sample, wherein each DNA first strand has a reverse common primer binding site. Is incorporated; a step of selecting each DNA first strand; a step of synthesizing at least one DNA second strand from each of the at least one DNA first strand, wherein each cDNA second strand is forwarded. A step of incorporating a common primer binding site; a step of selecting each cDNA second strand; and a step of amplifying the DNA strand using a common primer. Alternatively, the method may be performed using a gDNA template. The method described herein avoids primer dimer formation by selecting DNA strands and removing primers before amplification, and allows for much higher sensitivity and efficiency compared to conventional multiplex PCR methods.

  As used herein, “disease” means an infection, condition, or condition caused or associated with an agent.

  As used herein, a "disease factor", regardless of its form, includes, but is not limited to, a bacterium, cancer cell, virus, or parasite that contains a nucleic acid sequence and causes or contributes to a disease in a subject. Means any organism, including

  As used herein, "first primer mix" means a mixture comprising at least one reverse primer and / or at least one forward primer configured to bind to gDNA.

  As used herein, “forward primer mix” means a mixture that includes at least one forward primer.

  As used herein, “identifying marker” refers to a nucleotide sequence used as a label to identify a particular sample of mRNA or gDNA, a nucleic acid strand, or a single cell source.

  As used herein, "reverse primer mix" refers to a mixture that includes at least one reverse primer.

  As used herein, “sample” refers to a substance containing DNA or RNA.

  As used herein, "second primer mix" refers to a mixture comprising at least one reverse primer and / or at least one forward primer configured to bind to at least one DNA first strand. I do.

  As used herein, "subject" means a mammal, preferably a human.

  Conventional multiplex PCR methods include a selection step (i.e., removal of the primer mix or separation of DNA strands), but this is done only after the amplification product has been produced by PCR (e.g., arm-PCR [WO / 2009/124293], tem-PCR [WO / 2005/038039], each further requiring the use of nested primers). However, if selection is performed after PCR is complete, not only will the first few PCR cycles form primer dimers, but the primer dimers will compete with the target sequence for DNA polymerase activity throughout the PCR reaction. Applicants show that primer dimer also leads to a reduction in reaction sensitivity. Furthermore, since the amplification product and the primer dimer are similar in molecular weight and charge, separation becomes difficult. As shown in the examples below, in extreme cases, amplification of the primer dimer monopolizes the reaction and completely eliminates amplification of the target sequence. DNA polymerases are better at producing and binding shorter amplification products than at producing longer products, thus further accelerating the generation of primer dimers. Even when a primer dimer is formed but does not completely inhibit amplification of the target sequence, competition between the target sequence and the primer dimer reduces the amount of desired amplification product, which reduces the sensitivity of the overall reaction. And, when carried over to sequencing, produces a product that results in a loss of sequencing reads and unnecessary cost.

  Applicants argue that the current paradigm of multiplex optimization has inherent drawbacks. This is because a primer dimer can be formed even with an overlap of only 1 bp. Thus, prediction of primer dimers is not possible and needs to be addressed by new methodologies. Here we show that the efficiency of separating the desired DNA strand from the primer mix (referred to herein as "selection") is much better when the selection is performed after reverse transcription rather than after PCR. To When relatively long oligonucleotides are used, such as when creating a library compatible with next-generation sequencing (NGS), primer dimer formation may produce a product of approximately 200 bp in length. Separation is much more difficult and inefficient. However, bringing the selection step after reverse transcription results in the separation of the approximately 2 kb RNA: DNA duplex from the individual primers (typically less than 80 bp), which is much easier. Additional selection after second strand synthesis in the described method is also easier than post-PCR selection, but it does not form first-strand DNA: DNA duplex with shorter primers (80 bp ). In short, the sensitivity that is important in single-cell applications is already lost after waiting for the amplification product to separate or rescue from the primer dimer after PCR. In contrast, by preventing the reverse primer mix from interacting with the forward primer mix (eg, separating reverse transcription and second strand cDNA synthesis) or, in the case of gDNA, the first primer mix By preventing interaction with the mix and removing the reverse or forward primer mix before any PCR steps (or removing the first or second primer mix used in the case of gDNA) This eliminates the possibility of primer dimer formation and focuses the reaction energy on amplification of the desired product.

  As further support, we argue that single cells can be considered as dynamic templates with various gene expression patterns. In single cells, it is not possible to predict how the multiplex system will respond given the presence or absence of different amounts of template. Primer dimers that should not form in the presence of the template may form in the absence of the specific gene, making primer design impossible due to the diversity of expression at the single-cell level.

  To overcome these difficulties, Applicants have proposed a multi-step multiplex reverse transcription (RT) PCR of RNA or a multi-step multiplex of gDNA referred to herein as dimer avoidance multiplex polymerase chain reaction (dam-PCR). A methodology was developed to avoid primer dimers in plex PCR. The reverse transcription step is performed separately from both second strand DNA synthesis and PCR amplification using universal primers. Similarly, for gDNA, strand tagging with the first primer mix is performed separately from second strand tagging performed with the second primer mix. By selecting the synthesized DNA at each step and tagging the first-strand cDNA or gDNA with one cycle of tagging and extension, the tendency for primer-dimer formation is avoided and DNA polymerase activity is higher than for primer-dimers. Also concentrate on the amplification of the target of interest, and the amplification sensitivity is greatly increased. Exemplary embodiments of the disclosed method using an RNA template are shown in FIGS. 1A-1C, and exemplary embodiments of the disclosed method using a gDNA template are shown in FIGS. 2A-2C.

  During reverse transcription, one or more reverse primers containing binding sites that are 3 ′ gene specific and 5 ′ universal are used for first strand cDNA synthesis. One key to reducing primer dimer after reverse transcription is the very accurate selection of the first-strand cDNA: RNA complex ("first-strand: RNA complex"), and from this major product band Efficient removal of oligonucleotides of sequence length (typically less than 80 bp). This is particularly important when quantifying RNA by labeling each RNA with a separate oligonucleotide, as carryover of the uniquely labeled primer impairs the ability to accurately quantitate the labeled nucleic acid species. . After reverse transcription, a selection step is performed to remove unused reverse primer. This “selection” step separates DNA strands (eg, first strand cDNA: RNA complex in solution or single strand first strand cDNA) from the unused primer mix and removes the magnetic beads (eg, solid phase reversible). Actively select DNA strands by immobilization (SPRI) beads), by streptavidin-biotin beads, by enzymes, by columns, by gel purification, or by other physical, chemical or biochemical means. It is also possible to remove unused primers and any DNA polymerase. In the examples, Applicants have shown a selection efficiency of removing more than 99.99% of unused primer.

  When the first strand cDNA synthesis is completed and the reverse transcription primer is efficiently removed by selection of the first strand cDNA, second strand cDNA synthesis is carried out using a forward primer mix, which is referred to as second strand DNA tagging. ". One cycle of DNA polymerase activation and subsequent annealing is sufficient to tag the first strand cDNA product with the forward primer mix in the absence of any reverse primer. Since only one cycle is performed and unused primers are removed before PCR, individual nucleic acid species tags can also be introduced at this step. At this point, it is also possible to perform a limited number of cycles of linear amplification (primer annealing and extension and / or isothermal amplification in the absence of reverse primer) to increase yield. However, too many additional cycles of annealing and extension may increase the risk of detrimental primer dimer formation between primers in the forward mix and must be determined experimentally for a given multiplex system. Would. Applicants show that the first and second strand tagging can each be fully amplified in a single cycle, as evidenced by amplification from single cells. After the second strand DNA synthesis, a second selection step is performed to remove unused forward primer mix. This selection step separates the DNA strands from the unused primer mix and can be performed by magnetic beads (eg, SPRI beads), by streptavidin-biotin beads, by enzymes, by columns, by gel purification, or by other physical DNA strands can be actively selected by chemical, or biochemical means, or, conversely, unused primers and any DNA polymerase can be removed. The basic concept is the same as the first choice, that is, it eliminates the complexity of the primer and does not form a primer dimer that can defeat the desired main product.

  At this point, the “tagged” DNA contains a universal primer binding site at both the 5 ′ and 3 ′ ends. All tagging mixes of the reverse primer and the forward primer are removed by this stage. The universal site is generally the sequencing adapter or part of the adapter required for the selected NGS platform. However, any universal engineering design site is available. PCR is performed using a primer pair common to the universal "tagged" second strand cDNA. These primers complement the exponential amplification stage. Since primer complexity has been reduced to a two primer system (where the 3 'ends are not reverse complementary and the primer pairs have been screened for primer dimer formation), primer dimers are produced during this PCR. Tendency is eliminated.

  For low input doses, such as single cells, a second round of PCR may need to be performed. In that case, complete the first round of PCR selection as described above and perform a two-step PCR using the universal primer pair with the new enzyme, buffer, and dNTPs to concentrate the first round product I do. In each case, a final library selection is performed, and such libraries can be used directly for pooling, pre-sequencing QC, and sequencing using techniques known in the art.

  In some cases, gDNA is modified, but the same principle is applied as a whole, and the production of primer dimers is avoided. The first round of PCR is performed in a single cycle of denaturation and annealing steps using only one set of multiplexed primers (first primer mix). To increase sensitivity, linear or isothermal amplification can be performed as long as primer dimer formation in the mix is limited. The first strand: DNA complex is purified as described above and the second strand synthesis is performed in a single cycle using the second primer mix. Linear or isothermal amplification can be performed to increase sensitivity. After second strand synthesis, selection is performed as described above, and PCR is performed using the universal primer pair as described for the RNA process.

  The methods disclosed herein are useful for evaluating medical, environmental, food, and other samples to identify microorganisms and other factors in those samples, including viruses, bacteria, fungi, plants and And / or to detect the presence and relative abundance of nucleic acids from animal cells.

  One advantage of the methods described herein is that, unlike conventional multiplex PCR methods, primer dimer formation is avoided, thereby increasing the ability of the primer dimer to affect target sequence amplification. The selection of primer sets that would otherwise occur is greatly simplified. Another advantage of the methods described herein is that primer-dimer formation is avoided, resulting in multiplex PCR that is sensitive to single cells. RNA can be targeted at the single cell level using the methods of the present disclosure. In addition, the cost per read of each target sequence is greatly reduced by reducing the number of wasted reads that would otherwise be caused by the presence of primer dimers. Another advantage of the method, particularly with respect to the coverage of large gene segments of gDNA, is that data from duplicate amplification products can be generated without the introduction of harmful shorter amplification product byproducts that reduce sensitivity. is there. These by-products are inevitable when similar strategies are used in conventional multiplex PCR. This dam-PCR method allows for the strategic design of the first and second primer mix, allowing for longer coverage while limiting the interaction between specific primers producing short amplification products that compete for DNA polymerase activity I do.

Materials and Methods: Dam-PCR and arm-PCR Setup
Primers covering the T cell receptor alpha locus, beta locus, and additional phenotypic markers were multiplexed into the same mix or forward and reverse mix depending on the PCR strategy. The arm-PCR mix consisted of 218 forward primers and 68 reverse primers in the same mix, covering 247 or more targets. Although targets are defined in terms of reference sequences, the actual number of target sequences in a given sample is typically thousands in a bulk RNA sample due to the variability of the rearranged TCR loci. For single cells, depending on the cell phenotype, there may be 5 to 10 or more targets. For dam-PCR, the forward mix was processed separately from the reverse mix, removing the outer primers for the nested portion of the arm-PCR, for a total of 107 forward primers and 32 reverse primers. The inner primer containing the universal tag was common to both mixes and included a primer pair known to be responsible for primer dimer formation. In both primer sets, a portion of Illumina 2 Index Compatible Sequencing Primer B was ligated to each forward inner primer, a portion of Illumina 2 Index Sequencing Communal Primer A and a 6 nucleotide sample barcode. The sequence was ligated to the reverse inner primer. In certain experiments, an index of 20 random nucleotides tagging individual nucleic acid species was included adjacent to the sample barcode.

  In the arm-PCR approach, cDNA was reverse transcribed from total RNA samples using a nested primer mix of 286 primers and reagents from the OneStep RT-PCR kit (Qiagen, Valencia, CA). For arm-PCR, the first round of RT-PCR (referred to as “RT-PCR1” or “PCR1”) was performed as follows: 50 ° C. for 60 minutes; 95 ° C. for 15 minutes; 94 ° C. for 30 seconds. 10 cycles of 5 minutes at 60 ° C, 30 seconds at 72 ° C; 10 cycles of 30 seconds at 94 ° C, 3 minutes at 72 ° C; 5 minutes at 72 ° C, and hold at 4 ° C. Following RT-PCR1, one 0.7x SPRISelect bead selection (Beckman Coulter, Blair, CA) was performed once, and the nucleic acid product was eluted from the beads using a Promega Gotaq G2 Hotstart PCR mix (Promega, Madison, WI). A second round of PCR (referred to as "PCR2") was performed as follows using a set of communal primers complementary to the Illumina adapter sequence: 95 ° C for 3 minutes; 94 ° C for 30 seconds, 72 ° C at 72 ° C. 30 cycles of 90 seconds; 5 minutes at 72 ° C, and hold at 4 ° C.

  In dam-PCR, the reverse transcription step was performed using a Qiagen OneStep RT-PCR kit at 50 ° C. for 240 minutes in the presence of a reverse primer mix, and the 0.7x SPRI bead selection was performed twice. Strand cDNA was separated from the reverse primer mix. After reverse transcription, one cycle of tagging using Promega GoTaq G2 HotStart DNA polymerase and only forward primer mix (no reverse primer) in a Biorad C1000 thermocycler: 3 min initial denaturation and hot start at 95 ° C Annealing at 60-65 ° C., 3 min per 1 ° C. change; extension at 72 ° C. for 10 min and final hold at 4 ° C., or linear amplification strategy: initial denaturation and hot start 95 ° C. for 3 min, followed by 6 cycles of annealing Second strand DNA synthesis was performed by either annealing at 60 ° C. for 5 minutes, extending at 72 ° C. for 1 minute, and final hold at 4 ° C. After second strand DNA synthesis, the second strand DNA: DNA duplex was separated from the forward primer mix using two 0.7x SPRI bead selections. DNA products were eluted from the beads using a Promega Gotaq G2 Hotstart PCR mix containing a pair of primers common to the partial adapter sequence introduced in the reverse and forward priming steps. The cDNA was amplified with the universal primer pair in 20 cycles of PCR corresponding to the full cycle of the arm-PCR approach as follows: 95 ° C. for 3 minutes; 94 ° C. for 30 seconds, 72 ° C. for 6 minutes for 10 cycles. 10 cycles of 94 ° C for 30 seconds, 72 ° C for 3 minutes; Hold at 72 ° C for 5 minutes, and 4 ° C. In dam-PCR, one additional SPRI bead selection was performed as described above, and a second PCR was performed in the presence of new enzymes, buffers, and dNTPs as follows: 95 ° C. for 3 minutes ; 30 cycles of 94 ° C for 30 seconds, 72 ° C for 90 seconds; 5 minutes at 72 ° C, and hold at 4 ° C.

  After the second PCR reaction (ie, PCR2 in the case of arm-PCR), 10 μL of the PCR product was run on a 2.5% agarose gel to evaluate the success of amplification. Prior to sequencing of both arm-PCR and dam-PCR products, a library was selected using one 0.7x SPRI bead selection. The final library was eluted from the beads in 25 μL nuclease free water and measured on a Nanodrop (Thermoscientific, Carlsbad, CA). Equimolar amounts of each library were pooled for sequencing, except for libraries generated with primer dimer spike-ins. Libraries with spike-ins were pooled in half, due to library availability. The pooled library was quantified by Qubit quantification and evaluated by Bioanalyzer. The library was then quantified by Kappa qPCR, diluted to 8 pM with 10% PhiX spike-in, and sequenced using a 250 pair end read Illumina MiSeq v2, 500 cycle kit. A similar strategy was used for the described gDNA templates except that the reverse transcription step was omitted. Strategic primer design was used when designing the first and second primer mixes described herein to avoid production of short products.

Impact of Primer Dimer Formation on NGS Output: Loss of Sensitivity to Genes of Interest, Wasted Reads, and therefore Increased Sequencing Costs Optimized for traditional arm-PCR, as shown on the agarose gel in Figure 3A Single cells were amplified with the multiplex mix. Single cells may be isolated using techniques known in the art. arm-PCR is a nested multiplex RT PCR in which the product is `` saved '', for example, after RT-PCR, a small amount is extracted from the completed first amplification reaction to obtain an amplification product for the second amplification be able to.

  After amplification, the libraries representing each cell were pooled and subjected to an additional round of selection prior to sequencing with a 250 pair-end read Illumina MiSeq v2 500 cycle kit. The raw data was analyzed relative to successful target reads for evidence of primer dimers. The percentage of reads occupied by the primer dimer sequence was found to be inversely proportional to the band intensity of the main product band (FIG. 3B). For example, for sample BB-S73, the main product band was strong and the primer dimer accounted for only 1% of the number of sequencing reads in this cell, whereas for sample XH-S28, the number of product sequencing reads was relatively small. The primer dimer accounted for 89% of this cell data. In the data of the relatively strong product band such as the sample BB-S25, the primer dimer accounted for 25%.

  The advantage of a targeted sequencing approach compared to other methods, such as RNAseq, is that the sequencing depth required to cover the gene of interest is less, which means that the gene of interest This is because it is specifically targeted and amplified. When analyzing single cells, especially when treating each single cell as a sample, the cost can quickly become prohibitive if 25 times the number of reads is required to achieve the same coverage as the targeted seq approach. Analysis of primer dimer sequences in the described experiments showed that as much as 31% of the total sequencing reads were occupied by primer dimers, wasting valuable sequencing resources, and consequently Inappropriate costs as well as loss of sensitivity to cover different genes. However, obtaining amplification equivalent to the strongest amplification product band essentially eliminates waste of primer dimers, and has the added benefit of lower sequencing costs and increased sensitivity and coverage of the gene of interest. It is.

According to the NGS of the primer dimer, 1 bp duplication is sufficient for the formation of the primer dimer and cannot be eliminated by design alone. A primer-dimer is intentionally generated by performing arm-PCR using a multiplex mix in the presence (FIG. 4A), and the sequence of the obtained amplification product is determined by next-generation sequencing (NGS) technology. , Highlighting primer sequences that may need to be redesigned. Surprisingly, the data revealed over hundreds of different interacting pairs that produced dimerization products with only 1 bp duplication (FIG. 4B). In fact, the 1 bp overlapping primer dimer was one of the most frequently observed primer dimer pairs. Some potential pairs were predictable based on base pair complementarity and the 3 'end of each primer, but it was not possible to predict a 1 bp overlap, and therefore no related design So, again, the disadvantages of the current paradigm of multiplex PCR primer "optimization" became apparent (eg Canzar).

  However, the cloning of primer dimers and the sequencing of individual clones does not give a complete picture of the problem and the pure number of possible interactions. Until the sequence of the primer dimer band was physically observed by NGS, it was not clear that "design to overcome" the primer dimer was not possible. The previous results (FIG. 3A) show that there is still significant primer dimer production in the "successful" multiplex PCR. This primer dimer product competes with the band of interest throughout the PCR, greatly reducing PCR sensitivity and compromising the coverage of the gene of interest for the sample. Prior to NGS, the extent of the primer dimer effect could not be observed. Taken together, both of these results demonstrate that the alternative approach to PCR using dam-PCR disclosed herein is the only possible solution to the compatibility and sensitivity issues in multiplex PCR. ing.

Evidence of damage that a single primer pair can affect multiplex PCR From Figure 3A above, it can be seen that primer dimer formation can affect sequencing yields even when amplification is considered relatively `` successful ''. it is obvious. In other words, amplification resulted in a band of interest, but the sequencing results showed a significant amount of primer dimer along with the product of interest. However, Applicants can also show an example of the extreme case of a single primer pair that eliminates amplification of the desired product band in a multiplex PCR mix. As shown in FIG. 5, the addition of the T-bet forward primer to the mix containing the IL-10 primer eliminated the desired product band that was present prior to adding this one primer to the mix. Similarly, adding FoxP3 reverse primer to the mix of T-bet forward and reverse primer also resulted in loss of amplification of the main product band.

  Applicants have shown that one primer pair can eliminate amplification of the target of interest. In light of the present disclosure, which shows that primer dimers compete with the desired product for DNA polymerase activity during PCR, many multiplex PCR failures are attributed to (unwanted) "successful" primer dimer amplification products. This is a paradigm shift in understanding multiplex PCR. Having clarified this point, the present disclosure concludes that the best PCR approach is to compete for DNA polymerase activity between the main product band and the potential primer dimer as achieved by the dam-PCR methodology of the present disclosure. Is to avoid.

Increasing the annealing temperature is not enough to overcome the primer-dimer tendencyOne of the most frequently attempted methods to eliminate primer-dimer formation is to reduce the number of PCR cycles, and to reduce primer-dimer formation The concept of being inhibited by high temperatures is to raise the annealing temperature. When amplifying a single cell, reducing the number of cycles is not a viable option as long as the sensitivity is maintained, since many cycles are required to achieve amplification due to the low starting copy number. In FIG. 6, Applicants show that adjusting the annealing temperature to the point where the primers in the mix no longer bind to the target template is not sufficient to eliminate primer dimer formation and increase the annealing temperature. Alone does not eliminate the conflict. Primer pairs known to form primer dimers were tested at various annealing temperatures. If increasing the annealing temperature could reduce primer dimer production, increasing the temperature would have reduced the intensity of the primer dimer band. However, in each case, the band intensity of the primer dimer product was relatively unchanged despite the increase in the annealing temperature. At the highest allowable annealing temperature, there was only a slight reduction in two of the pairs tested.

Intentional spike-in of primer-dimer-prone primer pairs for comparison of arm-PCR and dam-PCR
Dam-PCR has the ability to overcome the inhibitory effects of primer dimers on amplification. Comparative experiments of arm-PCR and dam-PCR were performed using a multiplex mix containing 150 ng of RNA obtained from a mixture of CD3 + T cells and spleen. arm-PCR experiments are performed with both forward and reverse inner primers (outer primers do not make better comparisons) in the presence and absence of additional primer pairs known to cause primer dimer formation Performed by adding the mix. For dam-PCR, the same reverse primer mix used for arm-PCR amplification was added during the reverse transcription step. First strand cDNA was selected using magnetic beads and second strand tagging was performed using the same forward primer mix used in the arm-PCR experiments. A comparison between single cycle dam-PCR (one cycle of thermal denaturation, annealing and extension) and linear amplification dam-PCR (6 cycles of annealing and extension after thermal denaturation) was also performed. A tagged dam-PCR library was selected using magnetic beads, and the library was amplified in 20 cycles using a pair of communal primers. After tagging and one round of amplification with this common primer pair, 2 μL of the dam-PCR library was used for the transfer experiment from the first PCR amplification product to the second PCR reaction (FIG. 7B) and a similar transfer arm- Direct comparison with PCR test. The remaining dam-PCR libraries were selected using magnetic beads and amplified in the presence of new enzymes and buffers using the same universal primer pair for an additional 30 cycles. The total number of amplification cycles was equal to the arm-PCR protocol, including a 20-cycle amplification RT-PCR step and a 30-cycle second PCR.

  The results, shown in FIGS. 7A and 7B, clearly show that the dam-PCR strategy for primer dimer avoidance results in sensitive amplification despite the presence of a pair of primers that are likely to dimerize. Technically, there is no chance of dimerization in dam-PCR since the primer pair in question never comes into contact. Thus, the tagging step is performed in two independent steps, and amplification by PCR is actually performed (instead of a multiplex mix) with a pair of common primers. The multiplexed portion of the primers is only the forward set and reverse set (or first and second primer mix for gDNA), respectively. However, since each set, forward mix and reverse mix is used only once, potential primer-dimer pairs in the mix can never accumulate. All amplifications in FIGS. 7A and 7B include technical replicates. FIG. 7A shows bead selection between the first and second PCR reactions, and FIG. 7B shows 2 μL transfer between the first and second PCR reactions. As shown in FIGS. 7A and 7B, performing arm-PCR in the presence of a known harmful primer pair reduced the efficiency of amplification of the desired product. This effect is significant for the transfer between PCR1 and PCR2 in FIG. 7B, eliminating the main product band. Figures 7A and 7B show that for the same template RNA, the dam-PCR approach results in a very strong main product band and no production of primer dimers, especially with no chance of primer carryover as with 2 μL transfer At 7A, it is obvious. There is no apparent difference between a single cycle of dam-PCR on an agarose gel and the linear amplification approach, indicating that single cycle tagging is a viable approach.

Dam-PCR and single cell amplification Single cells are probably one of the most difficult templates. This is because the copy number of the target is low, and the interaction between the multiplex mix and the template becomes dynamic due to the phenotypic diversity of each cell. In the absence of template, certain primer pairs that do not show a high propensity to form primer dimers in collective bulk measurements may begin to dimerize due to lack of target. This adds to the complexity further, because of the inability to predict or design the interaction of the highly multiplexed primer mix with various low copy number templates. In Figure 8, Applicants show that, despite the diversity of gene expression at the single cell level, dam-PCR produced strong band intensity amplification products at the single cell level without primer dimer formation. Arm-PCR shows that the dynamic nature of single cells resulted in variable degrees of amplification of the target of interest, increasing primer dimer formation and smear, and clearly did not avoid primer dimer formation. Was. These negative side effects reduce the output of single cells as a result of the target of interest dropping out of the sequencing results, requiring the use of more sequencing reads per cell to provide sufficient gene coverage. However, expensive sequencing is performed for useless primer dimers, which ultimately significantly increases the cost of analyzing single cells.

Applicants conducted an experiment to demonstrate that the method disclosed herein would result in nearly 100% removal of unused primer between steps. Here, the applicants prepared a standard curve by serially diluting a 2 pmol stock solution of the reverse primer from 0.5% to 0. These dilutions were used as a basis to measure residual primer carryover from the two cleanup methods. These dilutions mimic the case where 0.5% to 0% carryover of the reverse primer occurs between reverse transcription and second strand synthesis. Forward primers with known primer-dimer propensity were added to each of the serial dilution mixes and the mixes were subjected to PCR to indirectly visualize the percentage of “remaining” reverse primer. Test samples included 2 pmol of reverse primer in each of the four tests (including technical replicates). These mixes were subjected to two different SPRI bead cleanup methods. To detect residual primers after cleanup, 2 pmol of the same forward primer used for the serially diluted sample was added to the selected product of the test sample. In this case, the residual reverse primer after cleaning was the only source of possible amplification. Test samples were subjected to the same PCR as the standard dilution curve. In this way, the band intensity of the amplification of the test sample can directly indicate the ratio of the residual reverse primer in the cleanup test by comparison with the standard curve shown in FIG. The results show that no residual primer was detected on the gel, so the CES selection had less than 0.01% carryover (or more than 99.99% removal), while the 0.7x SPRI selection had 0.10% carryover. (Or more than 99.9% removal).

Application of dam-PCR to gDNA gDNA was amplified by dam-PCR using three separate multiplex mixes covering different targets. One target mix covers the variable region of the human TCR beta locus, with the forward primer being the V gene segment and the reverse primer being the J region, allowing the determination of the VDJ rearrangement of the TCR beta locus from gDNA . The intron gap between the rearranged variable region and the C gene, coupled with sequencing length limitations, required the placement of a reverse primer at this position for gDNA coverage, and 13 sites to cover this gene segment. A reverse primer and 33 or more forward primers are required. For comparison, this mix was also used for the arm-PCR strategy. As shown in FIG. 10A, dam-PCR amplification produced an amplification product band when applied to gDNA, while arm-PCR generated smears, probably due to extra-site background amplification. When using the forward and reverse primer mix simultaneously (as in a typical multiplex PCR strategy), primers may bind to sites that have not been used for rearrangement, and products that compete with the rearrangement signal of interest May occur in the first few cycles. These extrasite reactions are reduced because dam-PCR allows only one cycle of tagging in both directions and selects only targets of primary interest between steps.

  To show that dam-PCR is effective for targets other than adaptive immune cells, multiplex mixes where mutations in cancerous malignancies cover common gene targets (tumor panel; FIG. A primer mix for detecting human leukocyte serotype (HLA type) was also applied to gDNA.

  With gDNA, there is more flexibility as to which primers to include in each mix. It is not necessary to include only the forward mix or just the reverse mix. Applicants therefore refer to the primer mix as the first mix and the second mix. This strategy can be applied in designing primer mixes that allow longer sequencing coverage with overlapping amplification products while reducing harmful background to both the amplification process and sequencing. Depending on the direction of first strand cDNA synthesis with RNA, a reverse mix must be used first. When gDNA is used as a template, the primer mix does not necessarily have to be hierarchized in the mix of the sense strand and the antisense strand. The primers can be used to design the overlap to facilitate subsequent bioinformatics reconstruction of larger gene products after sequencing. Usually, if all primer mixes can interact in the same mix, as in a typical multiplex PCR, off-target amplification will be produced, creating a background that competes with the product of interest, This creates a costly background in the sequencer, as shown in FIG. In dam-PCR, this primer mix strategy allows for targeted sequencing with reduced background when trying to cover larger gene targets. For the first primer mix, a pair of primers covering a much larger target can be used, as shown in FIG. Since each strand is processed independently, it will only be compatible with the respective first strand product when the next set of primers or second primer mix is applied to the second strand. Thus, this dam-PCR strategy can completely avoid primer interactions that could result in shorter useless products.

  Examples of both arm-PCR and dam-PCR applied to HLA targets from gDNA are provided in FIG. Lanes 1 and 2 show the amplification patterns when gene target A or target B was amplified using arm-PCR, respectively. Lane 3 shows the amplification pattern from the interaction of T2 forward and T1 reverse. Although this only shows a pattern, this product is an off-target product that could be produced if all four primers were multiplexed in the same PCR mix. Lane 4 shows the long amplification products of the T1 forward and T2 reverse primers. In this particular example, the product is also less desirable as it cannot be covered by the currently used NGS platforms due to sequence length limitations. However, any NGS platform that can cover longer products can sequence such products. Lanes 5-6 show arm-PCR from the complete multiplex mix. This result is mostly dominated by less desirable, short, off-target products. Target A only gene target (produced from T1 forward and T1 reverse), target B only gene target (produced from T2 forward and T2 reverse), and target A (produced from T1 forward and T2 reverse) Both gene targets, Target B and Target B, were smeared and almost absent due to the short product competing for DNA polymerase activity with the desired product. Lane 7 shows the amplification pattern when only gene target A was amplified using dam-PCR with T1 reverse during first cycle tagging and T1 forward during second cycle tagging. Lane 8 shows the amplification pattern when only gene target B was amplified using dam-PCR with T2 reverse during first cycle tagging and T2 forward during second cycle tagging. Lanes 9-10 show the results of a fully multiplexed dam-PCR strategy. For the first round of tagging with the first primer mix, T1 forward and T2 reverse were used to generate a longer first strand product. In the second round of tagging with the second primer mix, T1 reverse and T2 forward interacted independently with the first strand product generated during first strand tagging. After second-strand tagging, cleanup, and amplification using a pair of primers with a tag common to the first round of targets, there were no competing short products and amplification of the desired product was obtained. As evident from the gel images in lanes 9-10, the product band was the sum of the desired products displayed in lanes 1-2 or lanes 7-8. It is worth noting that single-cycle tagging is important. If the primers T1 forward and T2 reverse were subjected to additional cycles, as in normal PCR, a short competitor product may have been produced. However, these potentially harmful primers are removed in dam-PCR before performing any true amplification using the common universal primers.

  Reference to the singular should be understood to include the plural unless the context clearly dictates otherwise, or vice versa. A grammatical conjunction is intended to represent any disjunctive and connective combination of coordinating clauses, sentences, words, etc., unless otherwise indicated or clear from context. Thus, the term "or" should generally be understood to mean "and / or" and so on.

  Various aspects of the systems and methods described herein are exemplary. Various other aspects of the systems and methods described herein are possible.

Claims (24)

From mRNA containing at least one target sequence, reverse transcripting at least one cDNA first strand using a reverse primer mix to form at least one first strand cDNA, wherein the reverse primer mix comprises a reverse primer mix. Containing at least one reverse primer configured to incorporate a common primer binding site into each cDNA first strand;
Selecting each first strand cDNA;
From each of the at least one cDNA first strand, synthesizing at least one cDNA second strand using a forward primer mix, forming at least one first strand: second strand complex, The forward primer mix contains at least one forward primer, wherein each forward primer is configured to bind to a particular cDNA first strand and to incorporate a forward common primer binding site into each cDNA second strand. The process;
Selecting each first-strand: second-strand complex;
Amplifying the cDNA strand using a reverse common primer binding to the at least one reverse common primer binding site and using a forward common primer binding to the at least one forward common primer binding site; and A method of selecting a cDNA strand obtained. Amplifying the amplified cDNA strand using a reverse common primer that binds to the at least one reverse common primer binding site and using a forward common primer that binds to the at least one forward common primer binding site. 2. The method of claim 1, comprising:   2. The method of claim 1, wherein said reverse primer mix comprises at least one reverse primer, wherein said at least one reverse primer comprises additional nucleotides that are incorporated as identification markers into each first cDNA strand.   2. The method of claim 1, wherein said forward primer mix comprises at least one forward primer, wherein said at least one forward primer comprises additional nucleotides that are incorporated as identification markers into each second cDNA strand.   2. The method of claim 1, wherein each selection comprises separating the cDNA strand from the primer mix using magnetic beads.   2. The method of claim 1, wherein each selection comprises separating the cDNA strand from the primer mix by column purification.   2. The method of claim 1, wherein each selection comprises enzymatic cleavage of a primer mix.   2. The method of claim 1, wherein said first strand cDNA comprises a first strand cDNA: RNA complex. A method of diagnosing the presence of a disease in a subject, comprising the following steps:
Providing a sample from the subject, wherein the sample is suspected of containing a disease factor, wherein the disease factor is characterized by a target sequence;
Performing the method of claim 1 on nucleic acids in said sample;
Sequencing the amplified DNA strand; and detecting a target sequence from the disease factor. A method of generating an immune status profile for a subject, comprising the following steps:
Performing the method of claim 1 on nucleic acids from a sample of leukocytes from the subject;
Sequencing the amplified DNA strand;
Identifying and quantifying one or more DNA sequences representing T cell receptors, antibodies, and MHC rearrangements to create an immune status profile for said subject.   2. The method of claim 1, wherein said mRNA is obtained from a single cell. Synthesizing at least one first strand of DNA from genomic DNA containing at least one target sequence using a first primer mix to form a first strand: DNA complex, wherein the first primer mix Comprises at least one first primer, wherein each first primer is configured to bind to a particular target sequence and to incorporate a first common primer binding site into each DNA first strand. ;
Selecting each first strand: DNA complex;
Synthesizing at least one DNA second strand from each of the at least one DNA first strand using a second primer mix to form a first strand: second strand complex, The primer mix contains at least one second primer, wherein each second primer is configured to bind to a particular DNA first strand and to incorporate a second common primer binding site into each DNA second strand. The process;
Selecting each first-strand: second-strand complex;
Amplifying the DNA strand with a first common primer that binds to the at least one first common primer binding site and with a second common primer that binds to the at least one second common primer binding site; And selecting the amplified DNA strand. The first primer mix is a reverse primer mix, each first primer is a reverse primer, each first common primer is a reverse common primer, and each first common primer binding site is a reverse common primer binding site;
The second primer mix is a forward primer mix, each second primer is a forward primer, each second common primer is a forward common primer, and each second common primer binding site is a forward common primer binding site,
13. The method according to claim 12. The first primer mix is a forward primer mix, each first primer is a forward primer, each first common primer is a forward common primer, and each first common primer binding site is a forward common primer binding site;
The second primer mix is a reverse primer mix, each second primer is a reverse primer, each second common primer is a reverse common primer, and each second common primer binding site is a reverse common primer binding site.
13. The method according to claim 12. The first primer mix is a primer mix including at least one forward and at least one reverse primer, each first primer is a forward or reverse primer, and each first common primer is a forward or reverse common primer. And wherein each first common primer binding site is a forward or reverse common primer binding site;
The second primer mix includes at least one forward and at least one reverse primer, wherein none of the forward and reverse primers in the second primer mix are included in the first primer mix, The second common primer is a forward or reverse common primer, and each second common primer binding site is a forward or reverse common primer binding site.
13. The method according to claim 12.   Amplifying the amplified DNA strand using a reverse common primer that binds to the at least one reverse common primer binding site and using a forward common primer that binds to the at least one forward common primer binding site. 13. The method of claim 12, comprising.   13. The method of claim 12, wherein the primers in the first primer mix include additional nucleotides incorporated into each first DNA strand as an identification marker.   13. The method of claim 12, wherein the primers in the second primer mix include additional nucleotides incorporated into each second DNA strand as an identification marker.   13. The method of claim 12, wherein each selection comprises separating the DNA strand from the primer mix using magnetic beads.   13. The method of claim 12, wherein each selection comprises separating the DNA strand from the primer mix by column purification.   13. The method of claim 12, wherein each selection comprises enzymatic cleavage of a primer mix.   13. The method of claim 12, wherein said genomic DNA is obtained from a single cell. A method of diagnosing the presence of a disease in a subject, comprising the following steps:
Providing a sample from the subject, wherein the sample is suspected of containing a disease factor, wherein the disease factor is characterized by a target sequence;
Performing the method of claim 12 on nucleic acids in said sample;
Sequencing the amplified DNA strand; and detecting a target sequence from the disease factor. A method of generating an immune status profile for a subject, comprising the following steps:
13. Performing the method of claim 12 on nucleic acids from a sample of leukocytes from said subject;
Sequencing the amplified DNA strand;
Identifying and quantifying one or more DNA sequences representing T cell receptors, antibodies, and MHC rearrangements to create an immune status profile for said subject.

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